Agents for combating multiple drug resistance

ABSTRACT

Potentiating agents for combatting multiple drug resistance of susceptible tumor cells in a subject in need of such treatment are disclosed. The potentiating agents disclosed are triprolidine, cis-triprolidine, and pharmaceutically acceptable salts thereof.

FIELD OF THE INVENTION

The present invention relates to the use of triprolidine andtriprolidine-like compounds as adjuvant chemotherapy for neoplasiasresistant to multiple drugs. The present invention also relates to theuse of triprolidine-like compounds as an agent for enhancing thetherapeutic effect of multiple antitumor agents.

BACKGROUND OF THE INVENTION

Complete cures of various tumors like leukemias, lymphomas and solidtumors by the use of chemotherapeutic agents are rare because ofheterogeneous sensitivity of tumor cells to each antitumor agent. Cancerchemotherapy also fails because of intrinsic resistance of tumors tomultiple drug therapies. In other cases, a tumor may become resistant tothe antitumor agents used in a previous treatment. The therapeuticeffects of these agents are then eliminated. An even graver problem isthat recurrent cancers are resistant not only to the cancer suppressantsused in previous treatments, but also manifest resistance to otherantitumor agents, unrelated to the agent used previously either bychemical structure or by mechanism of action. These phenomenon arecollectively referred to as multiple drug resistance (mdr) andcontribute widely to cancer treatment failures in the clinic.

The major documented cause of multiple drug resistance is overexpressionof a membrane glycoprotein (the multiple drug transporter) responsiblefor pumping structurally diverse antitumor drugs from cells. See D.Houseman et al., A Molecular Genetic Approach to the Problem of DrugResistance in Chemotherapy, 504-517 (1987) (Academic Press, Inc.); R.Fine and B. Chabner, Multidrug Resistance, in Cancer Chemotherapy 8,117-128 (H. Pinedo and B. Chabner eds. 1986).

Tumor cells expressing elevated levels of the multiple drug transporteraccumulate far less antitumor agents intracellularly than tumor cellshaving low levels of this enzyme. The degree of resistance of certaintumor cells has been documented to correlate with both elevatedexpression of the drug transporter and reduced accumulation of antitumordrugs. See M. Gottesman and I. Pastan, J. Biol. Chem. 263, 12163 (1988);see also A. Fojo et al., Cancer Res. 45, 3002 (1985). This form ofmultiple drug cross-resistance involves agents derived from naturalproducts, such as the vinca alkaloids, the anthracyclines, theepipodophyllotoxins, actinomycin D and plicamycin. See I. Pastan and M.Gottesman, New England J. Med. 1388, 1389 Table 1 (May 28, 1987).

Adenocarcinomas derived from adrenal, kidney, liver, small intestine,and colon tissue are notorious for exhibiting inherent cross-resistanceto chemically unrelated chemotherapeutic agents. See M. Gottesman and I.Pastan, supra at 12165; see also A. Fojo et al., J. Clin. Oncol. 5, 1922(1987). These tissues normally express higher levels of the multidrugtransporter. Other tumors documented to express high levels of themultidrug transporter include pancreatic, carcinoid, chronic myelogenousleukemia in blast crisis, and non-small cell lung carcinoma. Tumorsdocumented to initially be drug-sensitive but to then become drugresistant include neuroblastoma, pheochromocytoma, acute lymphocyticleukemia in adults, acute nonlymphocytic leukemia in adults, nodularpoorly differentiated lymphoma, breast cancer and ovarian cancers. It isestimated by the National Cancer Institute that approximately half amillion tumor samples a year will be drug resistant because of aberrantlevels of expression of the multidrug transporter. See L. Goldstein etal., Expression of Multidrug Resistance Gene in Human Cancers, J.National Cancer Institute 81, 116 (1988).

Elevated levels of expression of the mdr drug transporter in thesetumors would lead to reduced intracellular levels of antitumor agents inthe tumor and would cause suppression of chemotherapeutic efficacy.Tumors having elevated levels of the multiple drug transporter wouldrequire therapeutic doses of cancer suppressants far in excess of tumorsexhibiting lower levels of the mdr drug transporter. Agents that inhibitthe active efflux of antitumor agents by the drug transporter or agentsthat potentiate the efficacy of chemotherapeutic agents would enhancethe activity of various antitumor agents on tumor cells. As a result ofthe present inventor's study, it has unexpectedly been found that whenthe agents disclosed herein are used together with an antitumor agent,they enhance the therapeutic effect of the antitumor agent.

A number of agents used clinically as calcium channel-blockers,calmodulin inhibitors and antiarrhythmic agents promote the activity ofantitumor agents against resistant tumor cells, see Tsuruo et al.,Cancer Res. 44, 4303 (1984); 43, 2267 (1983). Verapamil, caroverine,clomipramine, trifluoperazine, prenylamine, diltiazem, nicardipine, andquinidine enhance the activity of antitumor agents against resistantsublines of murine leukemia cells. Most agents potentiating the activityof antitumor agents are calcium antagonists, and the seriouscardiotoxicities that arise during treatment have limited their clinicalusefulness. While the inventor does not wish to be bound by any theoryof operation for the present invention, it is noted that the agentsdisclosed herein are not known to have calcium antagonism. They have,however, been found to elevate the intracellular concentration ofantineoplastic drugs in tumor cells overexpressing the multiple drugtransporter. Sensitization of drug resistant tumors and elevation ofintracellular antitumor drug concentrations probably occur by amechanism different from calcium antagonism.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an agent for enhancingthe therapeutic effect of an antineoplastic agent by administering to asubject harboring a tumor a compound of Formula (I) below(triprolidine), a compound of Formula (II) below (cis-triprolidine), ora pharmaceutically acceptable salt thereof (hereafter referred to as the"potentiating agent"). ##STR1##

Another aspect of the present invention is a method of inhibitingmultiple drug resistance in a subject in need of such treatment byadministering the subject a potentiating agent in an amount effective tocombat multiple drug resistance.

DETAILED DESCRIPTION OF THE INVENTION

Potentiating agents exemplary of the present invention include:

(A) Triprolidine; and

(B) Z-2-[3-(1-Pyrrolidinyl) -1- (4-tolyl)-1-propenyl]pyridinehydrochloride, or cis-Triprolidine. These compounds are known. See,e.g., R. Ison et al., J. Pharm. Pharmac. 25, 887 (1973).

A preferred category of multiple drug resistant tumor cells to betreated by the method of the present invention are multiple drugresistant cells characterized by the multidrug transporter--mediatedpumping of antineoplastic agents out of the tumor cells. The multidrugtransporter protein is described in M. Gottesman and I. Pastan, supra.Thus, tumor cells treated by the present invention are preferably thosecharacterized by (a) the expression of the multidrug transporter proteinat high levels, or (b) the ability to express the multidrug transporterprotein upon selection by an antineoplastic agent.

Exemplary of tumor cells which express the multidrug transporter at highlevels (intrinsically resistant cells) are adenocarcinoma cells,pancreatic tumor cells, carcinoid tumor cells, chronic myelogenousleukemia cells in blast crisis, and non-small cell lung carcinoma cells.

Exemplary of tumor cells having the ability to express the multidrugtransporter protein upon selection by an antineoplastic agent areneuroblastoma cells, pheochromocytoma cells, adult acute lymphocyticleukemia cells, adult acute nonlymphocytic leukemia cells, nodularpoorly differentiated lymphoma cells, breast cancer cells and ovariancancer cells.

A preferred group of tumor cells for treatment in the present inventionare the adenocarcinomas, including adenocarcinomas of adrenal, kidney,liver, small intestine and colon tissue, with kidney adenocarcinomacells particularly preferred.

Preferred antineoplastic agents for use in the present invention arethose to which multidrug transporter--mediated multiple drug resistantcells develop resistance. Exemplary of such antineoplastic agents arevinca alkaloids, epipodophyllotoxins, anthracycline antibiotics,actinomycin D, plicamycin, puromycin, gramicidin D, taxol, colchicine,cytochalasin B, emetine, maytansine, and amsacrine (or "mAMSA").Preferred are vinca alkaloids, epipodophyllotoxins, anthracycleneantibiotics, actinomycin D, and plicamyoin.

The vinca alkaloid class is described in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 1277-1280 (7th ed. 1985)(hereafter "Goodman and Gilman"). Exemplary of vinca alkaloids arevincristine, vinblastine, and vindesine.

The epipodophyllotoxin class is described in Goodman and Gilman, supraat 1280-1281. Exemplary of epipodophyllotoxins are etoposide, etoposideorthoquinone, and teniposide.

The anthracycline antibiotic class is described in Goodman and Gilman,supra at 1283-1285. Exemplary of anthracycline antibiotics aredaunorubicin, doxorubicin, mitoxantraone, and bisanthrene. Daunorubicinand doxorubicin are preferred.

Actinomycin D, also called Dactinomycin, is described in Goodman andGilman, supra at 1281-1283. Plicamycin, also called mithramycin, isdescribed in Goodman and Gilman, supra at 1287-1288.

Subjects to be treated by the method of the present invention includeboth human and animal (e.g., dog, cat, cow, horse) subjects, and arepreferably mammalian subjects.

The potentiating agent is administered in an amount effective to enhancethe efficacy of the antineoplastic agent. The potentiating agent ispreferably administered in a total amount per day of not more than about50 mg/kg body weight, more preferably not more than about 25 mg/kg, andmost preferably not more than about 5 mg/kg. With respect to minimumdose, the potentiating agent is preferably administered in a totalamount per day of at least about 0.01 mg/kg, more preferably at leastabout 0.1 mg/kg, and most preferably at least about 1 mg/kg. Thepotentiating agent may be administered once or several times a day.

As noted above, compounds of Formulas (I) and (II) may be administeredper se or in the form of a pharmaceutically acceptable salt. When usedin medicine, the salts of the compound of Formulas (I) and (II) shouldbe both pharmacologically and pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare the free active compound or pharmaceutically acceptable saltsthereof and are not excluded from the scope of this invention. Suchpharmacologically and pharmaceutically acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulphuric, nitric, phosphoric, maleic, salicylic,p-toluenesulfonic, tartaric, citric, methanesulphonic, formic, malonic,succinic, naphthalene-2-sulphonic and benzenesulphonic. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group. Thus, the present invention also providespharmaceutical formulations, both for veterinary and for human medicaluse, which comprise the potentiating agent together with one or morepharmaceutically acceptable carriers thereof and optionally any othertherapeutic ingredients. The carrier(s) must be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not unduly deleterious to the recipient thereof.

Pharmaceutical formulations of the present invention may optionallyinclude an antineoplastic agent, preferably an agent as described above.Such a formulation is useful for concurrently administering anantineoplastic agent and the potentiating agent in a method as describedabove.

The formulations include those suitable for oral, rectal, topical,nasal, ophthalmic or parenteral (including subcutaneous, intramuscularand intravenous) administration. Formulations suitable for oral andparenteral administration are preferred.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into desired formulations.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the potentiatingagent as a powder or granules; or a suspension in an aqueous liquor ornon-aqueous liquid such as a syrup, an elixir, an emulsion or a draught.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which is optionally mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent or dispersing agent. Molded tablets comprised of a mixture of thepowdered active compound with a suitable carrier may be made by moldingin a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose to which may also beadded any accessory ingredient(s). Such accessory ingredient(s) mayinclude flavorings, suitable preservatives, an agent to retardcrystallization of the sugar, and an agent to increase the solubility ofany other ingredient, such as a polyhydric alcohol, for example glycerolor sorbitol.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active compound, which ispreferably isotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of theactive compound with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the active compound dissolved or suspendedin one or more media such as mineral oil, petroleum, polyhydroxyalcohols or other bases used for topical pharmaceutical formulations.The addition of other accessory ingredients, vide infra, may bedesirable.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavoring agents, binders,disintegrants, surface active agents, thickeners, lubricants,preservatives (including antioxidants) and the like.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof. Temperatures are givenin degrees Celsius unless otherwise indicated.

EXAMPLE 1 In Vitro Cytotoxicity of Potentiating Agents in ChineseHamster Ovary Cells

Chinese hamster ovary (CHO) tissue culture cells were obtained from Dr.Vic Ling, Princess Margaret Hospital, Toronto, Canada. The parental cellline (AuxB1) and a multidrug resistant line (C5S32) having an amplifiedform of the MDR drug transport protein were plated into 96-wellmicrotitre culture dishes at 250 or 500 cells per well in minimalessential medium, type alpha, 10% fetal calf serum and incubated in 95%oxygen/5% carbon dioxide for 48 hours. After this period, the medium waschanged and one-half of the culture was treated with Actinomycin D (ActD) (0.01 μM for AuxB1 cells and 0.5 μM for C5S32 cells). C5S32 cells areabout 200-fold resistant to Actinomycin D compared to the parental AuxB1cell line. In addition to Act D some of the cultures also received adose of the potentiating agent at 1 and 10 μM. Thus, four conditionswere tested in each screening assay: untreated cells in medium alone,cells receiving Act D alone, cells incubated with the potentiating agentalone, and cells incubated with a combination of Act D and thepotentiating agent. Both the parental and mdr cell lines were treatedwith these four conditions simultaneously. Each experimental conditionreported below is based on the average absorbance from eight replicatesamples. The incubation with Act D and the test drug continued for 96additional hours, after which 0.5 mg/ml MTT dye was added to thecultures and allowed to incubate for three hours. The cells weresolubilized by addition of DMSO and the absorbance at 570 nm wasmonitored. The absorbance is directly related to the number of survivingcells in the culture dish.

In Table 1 below, the absorbance was normalized so that cytotoxicity ofthe potentiating agent could be evaluated. Untreated cultures were givena value of 1.00 and the cultures receiving 1 and 10 μM of thepotentiating agent are reported as a fraction of this value. To evaluatethe compounds for inducing synergism with Actinomycin D, the absorbancevalues of cultures receiving Act D alone were assigned a value of 1.00and cultures receiving the combination of Act D and potentiating agentAct D are reported as a fraction of this control. In most experiments,this concentration of Act D gave a reduction in cell number 10-20% belowthe value of completely untreated cultures.

                  TABLE 1                                                         ______________________________________                                        In Vitro Cytotoxicity of Potentiating                                         Agents in Chinese Hamster Ovary Cells                                                    Wildtype    Drug Resistant                                                    AUXBI       C5S32                                                  Compound Dose    0       +ACT D  0     +ACT D                                 ______________________________________                                        (A)       1 μM                                                                              1.00    1.00    0.83  0.80                                            10 μM                                                                              1.00    1.00    0.73  0.72                                   (B)       1 μM                                                                              0.96    0.78    0.70  0.82                                            10 μM                                                                              0.91    0.60    0.22  0.35                                   ______________________________________                                    

EXAMPLE 2 In Vitro Cytotoxicity of Potentiating Agents in Human KBEpidermoid Carcinoma Cells

The procedure for assaying the cytotoxicity of potentiating agents withhuman KB epidermoid carcinoma cells is essentially the same as the assayprocedure described above for use with Chinese hamster ovary cells. Inbrief, KB 3-1 (wt) and KB V-1 (mdr) cells are plated at 500 cells/wellin 96-well culture plates in Dulbecco's modified eagle medium,supplemented with 10% fetal calf serum. After 48 hours of incubation at37° C., the media is changed and cells are treated with actinomycin D at0.1 nM (3-1) or 20 nM (V-1). The test potentiating agent is introducedto one-half the untreated cultures and one-half the Act D treatedcultures at 1 and 10 μM. After 96 hours of additional incubation at 37°C., 0.5 mg/ml MTT dye is added, the cells are incubated for three hours,after which the cells are dissolved in DMSO, and the absorbance is thenread at 570 nm. The data is given in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        In Vitro Cytotoxicity of Potentiating Agents                                  in Human KB Epidermoid Carcinoma Cells                                                   Wildtype    Drug Resistant                                                    KB 3-1      KB V-1                                                 Compound Dose    0       +ACT D  0     +ACT D                                 ______________________________________                                        (A)       1 μM                                                                              1.00    1.00    0.83  0.94                                            10 μM                                                                              0.89    1.00    0.69  0.64                                   (B)       1 μM                                                                              1.00    1.00    1.00  1.00                                            10 μM                                                                              0.87    1.00    0.72  0.88                                   ______________________________________                                    

The foregoing examples are illustrative of the present invention, andare not to be taken as restrictive thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

That which is claimed is:
 1. A method of combating multiple drugresistance of susceptible tumor cells in a subject in need of suchtreatment, comprising administering to the subject a compound, in anamount effective to combat multiple drug resistance of tumor cellssensitive to said compound, wherein said compound is selected from thegroup consisting of triprolidine, cis-triprolidine, and pharmaceuticallyacceptable salts thereof.
 2. A method according to claim 1, wherein saidtumor cells are adenocarcinoma cells.